Friedreich ataxia patients are homozygous for abnormally expanded GAA triplet-repeats in the first intron of the FXN gene. The expanded GAA triplet-repeat sequence results in a deficiency of FXN transcript which is reversed via administration of histone deacetylase inhibitors, indicating that transcriptional silencing is at least partly due to an epigenetic abnormality. Our preliminary data show that Friedreich ataxia patients have heterochromatin formation involving the critical +1 nucleosome of the FXN gene, thus offering a potential mechanism for the transcriptional silencing. We found that heterochromatin formation at the +1 nucleosome and the ensuing deficiency of FXN transcript are associated with overexpression of a novel antisense transcript, FAST1 (FXN Antisense Transcript 1), that overlaps with the +1 nucleosome, and higher levels of a novel, promoter-associated microRNA, miR-FXN1. Given that both antisense RNAs and microRNAs are able to induce heterochromatin formation, leading to transcriptional silencing in mammalian cells, we hypothesize that loss-of-function of the FXN gene in FRDA is caused by RNA-induced heterochromatin formation and transcriptional gene silencing. We will test this hypothesis by investigating the potential roles of FAST1 and miR-FXN1 in inducing heterochromatin formation and transcriptional gene silencing of the FXN gene in Friedreich ataxia. Furthermore, we will test if the epigenetic defect seen in fibroblast cell lines from patients is also detected in Friedreich ataxia neurons derived from differentiation of induced pluripotent stem cells. Implication of FAST1 and / or miR-FXN1 in the pathogenesis of Friedreich ataxia would make them rational therapeutic targets to permit specific reactivation of the FXN gene. Indeed, we will also test the feasibility of reactivation of the FXN gene in Friedreich ataxia via targeted repression of FAST1 and miR-FXN1. This proposal will enhance our understanding of the pathogenesis of Friedreich ataxia, the most common inherited ataxia, and will potentially have important implications for the development of a specific therapy. PUBLIC HEALTH RELEVANCE: Patients with Friedreich ataxia, the most common inherited ataxia, have expanded GAA repeat sequences in their FXN genes. Preliminary results indicate that this leads to altered packaging of the FXN genes, turning off gene expression, and causing a deficiency of the essential protein frataxin. Our experiments are designed to determine the precise mechanism of this altered packaging in Friedreich ataxia, and to decipher methods to reverse it and restore normal gene function.